The present invention is directed to an improved method for detecting hybridized biomolecules, such as deoxyribonucleic acid (DNA), in solid phase hybridization. The improved method results in increased hybridization signal intensities in the analysis of biomolecules.
In the analysis of biomolecules using a solid phase, a plurality of probe biomolecules may be attached to a solid polymeric matrix or substrate in a pre-determined format referred to as an array. During hybridization, the probe biomolecules are subjected to treatment with complementary target biomolecules to form hybrid duplexes or complexes. Areas of the array where duplexes or complexes form are then detected by known detection techniques.
Known hybridization techniques used in solid-phase hybridization include the Southern blotting technique, also known as the forward-blot technique, and the reverse Southern blotting technique. The Southern blotting technique allows for the mapping of a position of a DNA sequence relative to restriction enzyme sites. In the Southern blotting technique, target biomolecules are initially attached to a solid support substrate, and then the target biomolecules are treated with probe biomolecules and undergo hybridization with the probe biomolecules.
In the reverse Southern blotting technique, pre-synthesized probe biomolecules are initially attached to a solid support substrate, and then the probe biomolecules are treated with target biomolecules and undergo hybridization with the target biomolecules. The reverse Southern blotting technique does not limit the number of target biomolecules that may be used and allows for a high throughput, that is, a large volume of data may be collected in a short period of time.
Under ideal conditions, immobilized probe biomolecules should be easily accessible for hybridization with target molecules. However, under less than ideal conditions, hybridization between the probe biomolecules and the target biomolecules is often inhibited or prevented because the probe biomolecules in the array are not available for interaction with the target biomolecules. This unavailability can occur when the probe biomolecules which are initially attached to the solid support surface form duplexes or complexes with themselves or self-hybridize upon themselves, by folding back on themselves to form hairpin loop secondary structures. Such secondary structures are referred to as intra-strand interactions. The unavailability of the probe biomolecules for hybridization with the target biomolecules can also occur when the probe biomolecules hybridize with adjacent probe biomolecules on the array surface to form secondary structure attachments referred to as inter-strand interactions. The presence of intra-strand and inter-strand secondary structures on the probe biomolecules retards the process of hybridization with target biomolecules.
A known method of enhancing hybridization of complementary polynucleotides in a buffered aqueous medium under hybridization conditions is disclosed in U.S. Pat. No. 5,512,436 to Stone. Such method is concerned with increasing the rate of hybridization and includes the use of a blocking agent.
There is a need for an improved method in solid phase array analysis for detection of biomolecules that results in an increased occurrence of hybridization between the probe biomolecules and the target biomolecules, that minimizes the formation of inter-strand and intra-strand interactions on the probe biomolecules, and that results in an increased hybridization signal intensity.